Heart disease is the predominant cause of disability and death in all industrialized nations. In the United States, it accounts for about 335 deaths per 100,000 individuals (approximately 40% of the total mortality) overshadowing cancer, which follows with 183 deaths per 100,000individuals. Four categories of heart disease account for about 85-90% of all cardiac-related deaths. These categories include: ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease, valvular disease, and congenital heart disease. Ischemic heart disease, in its various forms, accounts for about 60-75% of all deaths caused by heart disease. One of the factors that renders ischemic heart disease so devastating is the inability of the cardiac muscle cells to divide and repopulate areas of ischemic heart damage. As a result, cardiac cell loss as a result of injury or disease is irreversible.
Human to human heart transplants have become the most effective form of therapy for severe heart damage. Many transplant centers now have one-year survival rates exceeding 80-90% and five-year survival rates above 70% after cardiac transplantation. Infections, hypertension, and renal dysfunction caused by cyclosporin, rapidly progressive coronary atherosclerosis, and inmunosuppressant-related cancers have been major complications however. Heart transplantation, moreover, is limited by the scarcity of suitable donor organs. In addition to the difficulty in obtaining donor organs, the expense of heart transplantation prohibits its widespread application. Another unsolved problem is graft rejection. Foreign hearts and heart cells are poorly tolerated by the recipient and are rapidly destroyed by the immune system in the absence of immunosuppressive drugs. While immunosuppressive drugs may be used to prevent rejection, they also block desirable immune responses such as those against bacterial and viral infections, thereby placing the recipient at risk of infection. There is a clear need, therefore, to address the limitations of the current heart transplantation therapy as treatment for heart disease.
To overcome the current limitations of whole heart transplantation to treat heart disorders, the present invention provides cardiomyocytes, compositions including the cardiomyocytes, and methods for treating disorders characterized by insufficient cardiac function by administering the cardiomyocytes to subjects with such disorders. The cardiomyocytes of the present invention offer several advantages over whole heart transplantation to treat cardiac disorders. The cardiomyocytes are isolated from pigs, which provide a convenient, relatively inexpensive, and abundant source of cardiomyocytes. Moreover, in some instances, the cardiomyocytes of the invention can be modified such that rejection of the cardiomyocytes upon introduction into a xenogeneic recipient is inhibited, thereby eliminating the requirement for generalized suppression of the immune system.
Accordingly, the present invention pertains to an isolated porcine cardiomyocyte or an isolated population of porcine cardiomyocytes suitable for transplantation into a xenogeneic subject, particularly a human subject. In a preferred embodiment, the xenogeneic subject has a disorder characterized by insufficient cardiac function. Examples of such disorders include ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease (cor pulmonale), valvular disease, congenital heart disease, and any condition which leads to congestive heart failure. The porcine cardiomyocyte(s), in unmodified form, has at least one antigen on the cell surface which is capable of stimulating an immune response against the cardiomyocyte in a xenogeneic subject, for example, a human. The antigen on the surface of the porcine cardiomyocyte is altered to inhibit rejection of the cardiomyocyte when introduced into a xenogeneic subject. In one embodiment, the cardiomyocyte surface antigen which is altered is an MHC class I antigen. This MHC class I antigen can be contacted, prior to transplantation into a xenogeneic subject, with at least one anti-MHC class I antibody, or a fragment or derivative thereof, which binds to the MHC class I antigen on the cardiomyocyte surface but does not activate complement or induce lysis of the cardiomyocyte. One example of an anti-MHC class I antibody is an anti-MHC class I F(abxe2x80x2)2 fragment, such as an anti-MHC class I F(abxe2x80x2)2 fragment of a monoclonal antibody PT85. The present invention also pertains to compositions which include porcine cardiomyocytes and antibodies, antibody fragments, or derivatives, which bind an antigen on the surface of the porcine cardiomyocytes. These compositions can be inserted into a delivery device, e.g., a syringe, which facilitates the introduction of the cardiomyocytes into a subject. In addition, the porcine cardiomyocytes of the invention can be grown as a cell culture in a medium suitable to support the growth of the cells. In one embodiment, the cell culture includes a population of porcine cardiomyocytes in which at least about 30% of the cardiomyocytes express cardiac troponin and/or myosin. In another embodiment, the cell culture includes a population of porcine cardiomyocytes which contract synchronously.
Porcine cardiomyocytes which are suitable for transplantation into a xenogeneic subject can be obtained from both embryonic (i.e., fetal), newborn, and adult pigs. Preferred porcine cardiomyocytes are embryonic porcine cardiomyocytes obtained from an embryonic pig at a selected gestational age. The preferred gestational age of embryonic pigs from which to obtain cardiomyocytes suitable for transplantation into xenogeneic subjects, particularly humans, is between about day twenty (20) and birth of the pig. In other preferred embodiments, the cardiomyocytes are isolated between about day twenty (20) and about day eighty (80), more preferably between about day twenty (20) and about day sixty (60), yet more preferably between about day twenty (20) and about day fifty (50), still more preferably between about twenty (20) and about day forty (40), still further preferably between about day twenty (20) and about day thirty (30), and most preferably between about day twenty-five (25) and about day twenty-eight (28) of gestation.
The invention further pertains to an isolated porcine cardiomyocyte or an isolated population of cardiomyocytes obtained from a pig which is essentially free from organisms which are capable of transmitting infection or disease to a xenogeneic recipient, e.g., a human. Categories of pathogens from which the pig is free can include parasites, bacteria, mycoplasma, and viruses. In one embodiment, the pig from which the cardiomyocytes are isolated is free of the following organisms: Toxoplasma, eperythrozoon, brucella, listeria, mycobacterium TB, leptospirillum, haemophilus suis, M. Hyopneumonia, porcine respiratory reproductive syndrome, rabies, pseudorabies, parvovirus, encephalomyocarditus virus, swine vesicular disease, techen (Porcine polio virus), hemagglutinating encephalomyocarditus, suipoxvirus, swine influenza type A, adenovirus, transmissible gastroenteritis virus, bovine viral diarrhea, and vesicular stomatitis virus. The cardiomyocytes obtained from pathogen-free pigs can be modified as described herein to inhibit rejection of the cardiomyocytes upon introduction into a xenogeneic subject. Preferred cardiomyocyte ages are also described herein. The present invention also pertains to compositions which include porcine cardiomyocytes obtained from pathogen-free pigs and antibodies, antibody fragments, or derivatives, which bind an antigen on the surface of the porcine cardiomyocytes. These compositions can also be inserted into a delivery device, e.g., a syringe, which facilitates the introduction of the cardiomyocytes into a subject. In addition, the porcine cardiomyocytes obtained from pathogen-free pigs can be grown as a cell culture in a medium suitable to support the growth of the cells. In one embodiment, the cell culture includes a population of porcine cardiomyocytes in which at least about 30% of the cardiomyocytes express cardiac troponin and/or myosin. In another embodiment, the cell culture includes a population of porcine cardiomyocytes which contract synchronously.
Another aspect of the invention pertains to methods for treating disorders characterized by insufficient cardiac function, e.g., such as ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease (cor pulmonale), valvular disease, congenital heart disease, and any condition which leads to congestive heart failure, in a subject, particularly a human subject. These methods include administering to a subject having such a disorder, porcine cardiomyocytes of the present invention. In one embodiment, the porcine cardiomyocytes which can be administered to a subject having a cardiac disorder are porcine cardiomyocytes which, in unmodified form, have at least one antigen on the surface of the cardiomyocytes which is capable of stimulating an immune response against the cardiomyocytes in a xenogeneic subject, for example, a human. The antigen on the surface of the porcine cardiomyocytes is altered to inhibit rejection of the cardiomyocytes when introduced into a xenogeneic subject. Examples of cardiomyocyte surface antigens and methods of altering such antigens are described herein. Preferred cardiomyocyte ages are also described herein. In another embodiment, the porcine cardiomyocytes which can be administered to a subject having a disorder characterized by insufficient cardiac function are porcine cardiomyocytes which are obtained from a pig which is essentially free from organisms which are capable of transmitting infection or disease to a xenogeneic recipient, e.g., a human, of the cardiomyocytes. Pathogen-free pigs are described in detail herein. These cardiomyocytes can also be modified as described herein. Transplantation of the porcine cardiomyocytes can be accompanied by administration of an immunosuppressive agent, e.g., cyclosporin A, FK506, RS-61443, or a T cell antibody, to the subject.